Enrofloxacin Rapid Detection in Aquatic Foods: Based on DNA Aptamer Sensor.

Enrofloxacin (ENR) is widely used as a synthetic fluoroquinolone antibiotic for disease control in aquatic animals. ENR aptamers were screened in this study using the magnetic bead-SELEX method, and a graphene oxide fluorescent sensor was developed to detect the ENR residues in aquatic products. Firstly, ENR was conjugated to amino magnetic beads by amidation reaction, and then the aptamer sequences showing high affinity to ENR were screened step by step by using the SELEX screening method. Finally, after 10 rounds of SELEX screening, six candidate aptamers with high affinity were obtained. Among these, ENR-Apt 6 was selected based on its secondary structure features, high affinity (Kd = 35.08 nM), and high specificity to ENR. Furthermore, a fluorescent sensor was prepared using graphene oxide and ENR-Apt 6. The results showed that the linear range of the sensor could reach 600 nM (R2 = 0.986), while its optimal linear range was 1-400 nM (R2 = 0.991), with the lowest detection limit of 14.72 nM. The prepared sensor was successfully used for the detection of ENR in real samples, with a recovery range of 83.676-114.992% and a relative standard deviation < 10% for most of the samples.

Aptamer-Based Sensor for Rapid and Sensitive Detection of Ofloxacin in Meat Products.

Ofloxacin (OFL) is widely used in animal husbandry and aquaculture due to its low price and broad spectrum of bacterial inhibition, etc. However, it is difficult to degrade and is retained in animal-derived food products, which are hazardous to human health. In this study, a simple and efficient method was developed for the detection of OFL residues in meat products. OFL coupled with amino magnetic beads by an amination reaction was used as a stationary phase. Aptamer AWO-06, which showed high affinity and specificity for OFL, was screened using the exponential enrichment (SELEX) technique. A fluorescent biosensor was developed by using AWO-06 as a probe and graphene oxide (GO) as a quencher. The OFL detection results could be obtained within 6 min. The linear range was observed in the range of 10-300 nM of the OFL concentration, and the limit of the detection of the sensor was 0.61 nM. Furthermore, the biosensor was stored at room temperature for more than 2 months, and its performance did not change. The developed biosensor in this study is easy to operate and rapid in response, and it is suitable for on-site detection. This study provided a novel method for the detection of OFL residues in meat products.

The role and mechanisms of PD-L1 in immune evasion during Talaromyces marneffei infection.

Talaromycosis, caused by Talaromyces marneffei (T. marneffei), is a systemic fungal disease that involves dissemination throughout the body. The ability of T. marneffei to evade the immune system is considered a crucial factor in its persistent infection, although the specific mechanisms are not yet fully understood. This study aims to investigate the molecular mechanisms underlying the occurrence of latent T. marneffei infection and immune evasion. The gene expression profile analysis in T. marneffei-infected mouse revealed that Pd-l1 exhibited the highest correlation strength with other hub genes, with a median of 0.60 (IQR: 0.50-0.69). T. marneffei infection upregulated the expression of PD-1 and PD-L1 in PBMCs from HIV patients, which was also observed in the T. marneffei-infected mouse and macrophage models. Treatment with a PD-L1 inhibitor significantly reduced fungal burden in the liver and spleen tissues of infected mice and in the kupffer-CTLL-2 co-culture system. PD-L1 inhibitor treatment increased CTLL-2 cell proliferation and downregulated the expression of PD-1, SHP-2, and p-SHP-2, indicating the activation of T cell viability and T cell receptor signaling pathway. Additionally, treatment with a PI3K inhibitor downregulated PD-L1 in T. marneffei-infected kupffer cells. Similar results were observed with treatment using the T. marneffei cell wall virulence factor ß-glucan. Overall, T. marneffei infection upregulated PD-L1 expression in HIV / T. marneffei patients, mice, and kupffer cells. Treatment with a PD-L1 inhibitor significantly reduced fungal burden, while activating T cell activity and proliferation, thereby promoting fungal clearance. Furthermore, the PI3K signaling pathway may be involved in the regulation of PD-L1 by T. marneffei.

Isolation and Characterization of Bacteriophage VA5 against Vibrio alginolyticus.

Bacteriophages, or phages, can be used as natural biological control agents to eliminate pathogenic bacteria during aquatic product cultivation. Samples were collected from seafood aquaculture water and aquaculture environmental sewage, and phage VA5 was isolated using the double-layer agar plate method, with Vibrio alginolyticus as the host bacteria. The purified phage strain was subjected to genome sequencing analysis and morphological observation. The optimal multiplicity of infection (MOI), the one-step growth curve, temperature stability, and pH stability were analyzed. Phage VA5 was observed to have a long tail. Whole-genome sequencing revealed that the genome was circular dsDNA, with 35,866 bp length and 46% G+C content. The optimal MOI was 1, the incubation period was 20 min, the outbreak period was 30 min, and the cleavage amount was 92.26 PFU/cell. The phage showed good activity at -20 °C, 70 °C, and pH 2-10. Moreover, the phage VA5 exhibited significant inhibitory effects on V. alginolyticus-infected shrimp culture. The isolated phage VA5 has a wide range of host bacteria and is a good candidate for biological control of pathogenic bacteria.

Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing.

Talaromyces marneffei (T. marneffei) immune escape is essential in the pathogenesis of talaromycosis. It is currently known that T. marneffei achieves immune escape through various strategies. However, the role of cellular alternative splicing (AS) in immune escape remains unclear. Here, we depict the AS landscape in macrophages upon T. marneffei infection via high-throughput RNA sequencing and detect a truncated protein of NCOR2 / SMRT, named NCOR2-013, which is significantly upregulated after T. marneffei infection. Mechanistic analysis indicates that NCOR2-013 forms a co-repression complex with TBL1XR1 / TBLR1 and HDAC3, thereby inhibiting JunB-mediated transcriptional activation of pro-inflammatory cytokines via the inhibition of histone acetylation. Furthermore, we identify TUT1 as the AS regulator that regulates NCOR2-013 production and promotes T. marneffei immune evasion. Collectively, these findings indicate that T. marneffei escapes macrophage killing through TUT1-mediated alternative splicing of NCOR2 / SMRT, providing insight into the molecular mechanisms of T. marneffei immune evasion and potential targets for talaromycosis therapy.

Construction of a murine model of latent infection and reactivation induced by Talaromyces marneffei.

OBJECTIVE: To establish a murine model of Talaromyces marneffei (T. marneffei) latent infection and reactivation, providing a foundation for exploring the molecular mechanisms underlying disease relapse. METHODS: BALB/c mice were tail vein injected with T. marneffei at 0 days post-infection (dpi) and treated with cyclophosphamide (CTX) intraperitoneally every four days, starting from 21 dpi or 42 dpi. Mice were observed for body weight changes, liver and spleen indices, histological characteristics of liver and spleen, fungal load detection in liver and spleen, and Mp1p qualitation in liver and spleen to assess T. marneffei infection severity. RESULTS: T. marneffei-infected mice exhibited a trend of initial weight loss followed by recovery and a subsequent decrease in weight after CTX injection throughout the observation period. Liver and spleen indices, as well as tissue damage, significantly increased during infection but later returned to normal levels, with a gradual rise observed after immunosuppression. Fungal load analysis revealed positive T. marneffei cultures in the liver and spleen at 7 dpi and 14 dpi, followed by negative T. marneffei cultures from 21 dpi until day 21 post-immunosuppression (42 dpi or 63 dpi); however, the spleen remained T. marneffei-cultured negative, consistent with the trend observed in Mp1p detection results. CONCLUSION: A latent infection and reactivation model of T. marneffei in mice was successfully established, with the liver likely serving as a key site for latent T. marneffei.

An efficient DNAzyme for the fluorescence detection of Vibrio cholerae.

Vibrio cholerae (Vc) causes cholera disease. Vc contamination is widely found in water and aquatic products, and therefore is a serious food safety concern, especially for the seafood industry. In this paper, we attempted the rapid detection of V. cholerae. Nine rounds of in vitro selection using an unmodified DNA library were successfully performed to find specific DNAzymes of Vc. Their activity was evaluated based on a fluorescence assay and gel electrophoresis. Finally, a DNAzyme (named DVc1) with good activity and specificity with a detection limit of 7.2 × 103 CFU/mL of Vc was selected. A simple biosensor was constructed by immobilizing DVc1 and its substrate in shallow circular wells of a 96-well plate using pullulan polysaccharide and trehalose. When the crude extracellular mixture of Vc was added to the detection wells, the fluorescent signal was observed within 20 min. The sensor effectively detected Vc in aquatic products indicating its simplicity and efficiency. This sensitive DNAzyme sensor can be a rapid onsite Vc detection tool.

Application of deep learning algorithm in the recognition of cryptococcosis and talaromycosis skin lesions.

BACKGROUND: Cryptococcosis and talaromycosis are known as 'neglected epidemics' due to their high case fatality rates and low concern. Clinically, the skin lesions of the two fungal diseases are similar and easily misdiagnosed. Therefore, this study aims to develop an algorithm to identify cryptococcosis/talaromycosis skin lesions. METHODS: Skin images of tararomiasis and cryptococcosis were collected from published articles and augmented using the Python Imaging Library (PIL). Then, five deep artificial intelligence models, VGG19, MobileNet, InceptionV3, Incept ResNetV2 and DenseNet201, were developed based on the collected datasets using transfer learning technology. Finally, the performance of the models was evaluated using sensitivity, specificity, F1 score, accuracy, AUC and ROC curve. RESULTS: In total, 159 articles (79 for cryptococcosis and 80 for talaromycosis), including 101 cryptococcosis skin lesion images and 133 talaromycosis skin lesion images, were collected for further mode construction. Five methods showed good performance for prediction but did not yield satisfactory results for all cases. Among them, DenseNet201 performed best in the validation set, followed by InceptionV3. However, InceptionV3 showed the highest sensitivity, accuracy, F1 score and AUC values in the training set, followed by DenseNet201. The specificity of DenseNet201 in the training set is better than that of InceptionV3. CONCLUSIONS: DenseNet201 and InceptionV3 are equivalent to the optimal model in these conditions and can be used in clinical settings as decision support tools for the identification and classification of skin lesions of cryptococcus/talaromycosis.

Time series prediction for the epidemic trends of monkeypox using the ARIMA, exponential smoothing, GM (1, 1) and LSTM deep learning methods.

Monkeypox is a critical public health emergency with international implications. Few confirmed monkeypox cases had previously been reported outside endemic countries. However, since May 2022, the number of monkeypox infections has increased exponentially in non-endemic countries, especially in North America and Europe. The objective of this study was to develop optimal models for predicting daily cumulative confirmed monkeypox cases to help improve public health strategies. Autoregressive integrated moving average (ARIMA), exponential smoothing, long short-term memory (LSTM) and GM (1, 1) models were employed to fit the cumulative cases in the world, the USA, Spain, Germany, the UK and France. Performance was evaluated by minimum mean absolute percentage error (MAPE), among other metrics. The ARIMA (2, 2, 1) model performed best on the global monkeypox dataset, with a MAPE value of 0.040, while ARIMA (2, 2, 3) performed the best on the USA and French datasets, with MAPE values of 0.164 and 0.043, respectively. The exponential smoothing model showed superior performance on the Spanish, German and UK datasets, with MAPE values of 0.043, 0.015 and 0.021, respectively. In conclusion, an appropriate model should be selected according to the local epidemic characteristics, which is crucial for monitoring the monkeypox epidemic. Monkeypox epidemics remain severe, especially in North America and Europe, e.g. in the USA and Spain. The development of a comprehensive, evidence-based scientific programme at all levels is critical to controlling the spread of monkeypox infection.

Maxent modeling for predicting the global potential distribution of talaromycosis marneffei / 中国热带医学

@#Abstract: Objective　To predict the potential distribution of talaromycosis marneffei (TSM) and analyze its driving factors, so as to provide evidence for the surveillance and prevention of this disease. Methods　The data of all laboratory-confirmed, non-duplicating TSM published in the English and Chinese literature from the first case in January 1964 to December 2018 was collected. A Maxent ecology model using environmental variables, Rhizomys distribution and HIV/AIDS epidemic was developed to forecast ecological niche of TSM worldwide, as well as identify the driving factors. Results　A total of 705 articles (477 in Chinese and 228 in English) were obtained during the study period. After excluding imported cases, a total of 100 foci information were included in the model. The area under the receiver operating characteristic (ROC) curve (AUC) of the model was 0.997 for the training set and 0.991 for the test set. Maxent model revealed that Rhizomys distribution, mean temperature of warmest quarter, precipitation of wettest month, HIV/AIDS epidemic and mean temperature of driest quarter were the top 5 important variables affecting TSM distribution. In addition to identifying traditional TSM endemic areas (South of the Yangtze River in China, Southeast Asian, North and Northeast India), other potential endemic areas were also identified, including parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Conclusion　Our finding has discovered hidden high-risk areas and provided insights about driving factors of TSM distribution, which will help inform surveillance strategies and improve the effectiveness of public health interventions against TM infections.

On-site detection of fish furunculosis by combining DNAzyme and carboxyl-functionalized graphene.

Furunculosis, which is caused by Aeromonas salmonicida, can induce septicemia, leading to the rapid death of fishes belonging to Salmonidae, Cyprinidae, and Fuscheridae, and lamprey. Targeting A. salmonicida, five DNAzyme sequences with the highest enrichment rates were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The enrichment rates were 34.78, 23.60, 8.91, 2.89, and 2.34%, respectively. The DNAzyme with the highest activity, named D-AS-2, showed specificity and sensitivity. D-AS-2 was combined with carboxyl-functionalized graphene to construct a biosensor, which showed good fluorescence response to scabies lesion samples. The diagnostic procedure was completed in <2 min and can be used for the on-site diagnosis of fish diseases. A low-cost, rapid, simple, and highly specific biosensor for the diagnosis of furunculosis was established based on DNAzyme and carboxyl-functionalized graphene.

Three Novel C-Repeat Binding Factor Genes of Dimocarpus longan Regulate Cold Stress Response in Arabidopsis.

Longan (Dimocarpus longan) is a typical southern subtropical fruit tree species that is sensitive to cold stress. C-repeat binding factors (CBFs), as transcription factors, are crucial components involved in the molecular regulation of the plant response to cold stress. However, the role of CBF homologs in the cold response regulation of longan remains largely unknown. Here, three novel CBF genes, DlCBF1, DlCBF2, and DlCBF3, were cloned from longan. DlCBF1 and DlCBF2 contain an AP2 domain and PKKPAGR and DSAWR CBF signature motifs, while DlCBF3 has mutations within these conserved signature motifs. DlCBF1/2/3 were mainly localized in the nucleus and specifically bound to CRT/DRE cis-elements, resulting in strong transcriptional activation. DlCBF1/2 exhibited tissue expression specificity, and their expression was induced by low temperature, while DlCBF3 had no tissue specificity and barely responded to low temperature. DlCBF1, DlCBF2, and DlCBF3 overexpression in Arabidopsis-enhanced cold tolerance by increasing proline accumulation and reducing reactive oxygen species (ROS) content, accompanied by upregulated expression of cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1) in the CBF cold stress response signaling pathway. In conclusion, the biological functions of DlCBF1/2/3 were somewhat conserved, but slow expression of DlCBF1/2 and low expression of DlCBF3 may partly cause the cold sensitivity of longan. Collectively, these results indicated that differences exist in the expression and function of CBF orthologs in the cold-sensitive plant species longan, and these findings may help to improve the understanding of the cold response regulation mechanism and provide important theoretical support for cold-tolerant breeding of longan.

FGF2 promotes metastasis of uveal melanoma cells via store-operated calcium entry.

Uveal melanoma (UM), the most common primary intraocular malignancy in adults, is highly metastatic and associated with dismal prognosis. Fibroblast growth factor 2 (FGF2) has been shown to induce cell proliferation and angiogenesis of melanoma and other malignancies. However, the expression of FGF2 in UM and its effects on melanoma cell migration are not well known. In this study, we found FGF2 expression was related to UM histological subtype and presence of metastasis. In vitro experiments showed that FGF2 treatment caused increased horizontal and vertical migration and F-actin cytoskeleton assembly as well as decreased adhesive activity of MUM2B cells, together with increased intracellular calcium concentration and expression of ORAI1 and STIM1 - two key regulatory proteins of store-operated calcium entry (SOCE). The mouse xenograft model showed that MUM2B cells with FGF2 stimulation grew into larger tumor masses and were prone to metastasis. In addition, the SOCE inhibitor 2-aminoethoxydiphenyl borate (2-APB) reversed all of these effects of FGF2. Finally, human UM samples and mouse xenograft model samples were used to confirm the correlation of FGF2 with ORAI1 and STIM1 expression. Taken together, our study suggests that FGF2 promotes metastasis of UM via SOCE.

Dickkopf-1 inhibits Wnt3a-induced migration and epithelial-mesenchymal transition of human lens epithelial cells.

Posterior capsular opacification (PCO) is a major post-operative complication of cataract surgery. Epithelial-mesenchymal transition (EMT) contributes to PCO. We previously indicated that Wnt3a induces the EMT of human lens epithelial cells (LECs) and plays an important role in the development of PCO. The present study aimed to test the potential effect of Dickkopf-1 (Dkk1) on Wnt3a-induced cell migration and the EMT of LECs and to explore possible cellular mechanisms. The secretion of Dkk1 was reduced in the rabbit PCO model, and Dkk1 injected into the eyes post-surgical manipulation prevented PCO formation. Cultured HLE-B3 cells were then transfected with Wnt3a in the presence or absence of Dkk1. Dkk1 treatment restored the epithelial phenotype and reversed the expression of EMT-associated proteins induced by Wnt3a. Dkk1 suppressed LEC migration and the expression of matrix metalloproteinase-1 (MMP-1), and the activity of MMP-2 and MMP-9. Dkk1 inhibited the nuclear accumulation of ß-catenin, which is the key regulator of the canonical Wnt signaling. Our results indicate that Dkk1 inhibits Wnt3a-induced migration and the EMT of human LECs.The results contribute to the prevention of PCO formation and development.

Identifying Litchi (Litchi chinensis Sonn.) Cultivars and Their Genetic Relationships Using Single Nucleotide Polymorphism (SNP) Markers.

Litchi is an important fruit tree in tropical and subtropical areas of the world. However, there is widespread confusion regarding litchi cultivar nomenclature and detailed information of genetic relationships among litchi germplasm is unclear. In the present study, the potential of single nucleotide polymorphism (SNP) for the identification of 96 representative litchi accessions and their genetic relationships in China was evaluated using 155 SNPs that were evenly spaced across litchi genome. Ninety SNPs with minor allele frequencies above 0.05 and a good genotyping success rate were used for further analysis. A relatively high level of genetic variation was observed among litchi accessions, as quantified by the expected heterozygosity (He = 0.305). The SNP based multilocus matching identified two synonymous groups, 'Heiye' and 'Wuye', and 'Chengtuo' and 'Baitangli 1'. A subset of 14 SNPs was sufficient to distinguish all the non-redundant litchi genotypes, and these SNPs were proven to be highly stable by repeated analyses of a selected group of cultivars. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis divided the litchi accessions analyzed into four main groups, which corresponded to the traits of extremely early-maturing, early-maturing, middle-maturing, and late-maturing, indicating that the fruit maturation period should be considered as the primary criterion for litchi taxonomy. Two subpopulations were detected among litchi accessions by STRUCTURE analysis, and accessions with extremely early- and late-maturing traits showed membership coefficients above 0.99 for Cluster 1 and Cluster 2, respectively. Accessions with early- and middle-maturing traits were identified as admixture forms with varying levels of membership shared between the two clusters, indicating their hybrid origin during litchi domestication. The results of this study will benefit litchi germplasm conservation programs and facilitate maximum genetic gains in litchi breeding programs.

A self-assembled system for tumor-targeted co-delivery of drug and gene.

A new cationic polymer eprosartan-graft-PEI (ESP) containing eprosartan (ES) and polyethylenimine 1.8K was successfully developed and employed as a safe gene vector to assemble a drug (ES) and gene co-delivery complex (ESP/pDNA). Chondroitin sulfate (CS) was then used as a coating polymer to shield the surface charge of ESP/pDNA complexes, as well as a tumor targeting entity to ensure specific delivery of CS/ESP/pDNA complexes. The CS/ESP/pDNA complexes with desirable particle size and zeta potential, exhibited amidase-responsive drug release and CS-mediated endocytosis in vitro. As compared with ESP/pDNA complexes, in vivo imaging results demonstrated decreased reticuloendothelial system uptake and remarkably increased tumor accumulation of CS/ESP/pDNA complexes. All these findings indicated the potential of CS/ESP/pDNA as a promising tumor-targeted drug and gene co-delivery system.

Versatile reticular polyethylenimine derivative-mediated targeted drug and gene codelivery for tumor therapy.

The study is aimed to develop a versatile reticular polyethylenimine (PEI) derivative eprosartan-g-PEI (ESP) conjugate-mediated targeted drug and gene codelivery system for tumor therapy. Eprosartan (ES), an angiotensin II type 1 receptor blocker (ARB), which has been proven to exert beneficial effects on tumor progression, vascularization, and metastasis as the conventional antihypertensive drug, was conjugated with PEI-1.8K chains into ESP via a bis-amide bond of pH-sensitivity to overcome high cytotoxicity and nontargeted gene delivery of PEI-25K. P53 gene was encapsulated in the ESP to form the codelivery system of ESP/p53 complexes, and this system was comprehensively characterized. In vitro ESP/p53 complexes had a significant effect on inhibiting angiogenesis by reducing the expression and secretion of VEGF. In vivo the effective antitumor activity of ESP/p53 complexes was observed on nude mice bearing PANC-1 xenografts, and the microvessel density (MVD) examination demonstrated that ESP/p53 complex-produced antitumor efficacy was closely correlated with the efficient angiogenesis repression. These findings disclosed that the multifunctional ESP/p53 complexes might be a promising dual anticancer drug and gene codelivery system.

A chitosan-graft-PEI-candesartan conjugate for targeted co-delivery of drug and gene in anti-angiogenesis cancer therapy.

A multifunctional copolymer-anticancer conjugate chitosan-graft-polyethyleneimine-candesartan (CPC) containing low molecular weight chitosan (CS) backbone and polyethyleneimine (PEI) arms with candesartan (CD) conjugated via an amide bond was fabricated as a targeted co-delivery nanovector of drug and gene for potential cancer therapy. Here, CD was utilized to specifically bind to overexpressed angiotensin II type 1 receptor (AT1R) of tumor cells, strengthen endosomal buffering capacity of CPC and suppress tumor angiogenesis. The self-assembled CPC/pDNA complexes exhibited desirable and homogenous particle size, moderate positive charges, superior stability, and efficient release of drug and gene in vitro. Flow cytometry and confocal laser scanning microscopy analyses confirmed that CD-targeted function and CD-enhanced buffering capacity induced high transfection, specific cellular uptake and efficient intracellular delivery of CPC/pDNA complexes in AT1R-overexpressed PANC-1 cells. In addition, CPC/wt-p53 complexes co-delivering CD and wild type p53 (wt-p53) gene achieved synergistic angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor (VEGF) mRNA and protein via different pathways in vitro, as compared to mono-delivery and mixed-delivery systems. In vivo investigation on nude mice bearing PANC-1 tumor xenografts revealed that CPC/wt-p53 complexes possessed high tumor-targeting capacity and strong anti-tumor activity. Additional analysis of microvessel density (MVD) demonstrated that CPC/wt-p53 complexes significantly inhibited tumor-associated angiogenesis. These findings suggested that CPC could be an ideal tumor-targeting nanovector for simultaneous transfer of drug and gene, and a multifunctional CPC/wt-p53 co-delivery system with tumor-specific targetability, enhanced endosomal buffering capacity and synergistic anti-angiogenesis efficacy might be a new promising strategy for effective tumor therapy.

Direct cytosolic siRNA delivery by reconstituted high density lipoprotein for target-specific therapy of tumor angiogenesis.

We described here the mechanisms by which small interfering RNA (siRNA) molecules incorporated in reconstituted high density lipoprotein (rHDL) were efficiently transferred into the cytoplasm of cells to perform target-specific therapy of tumor angiogenesis. Using fluorescent-tagged apolipoprotein A-I (apoA-I) and cholesterol-conjugated siRNA (Chol-siRNA), it was confirmed with FACS and confocal microscopic measurements that Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were successfully established and apoA-I certainly was attached to the surface of Chol-siRNA-loaded lipoplexes (Lipos/Chol-siRNA complexes). Stably assembled rHDL/Chol-siRNA complexes demonstrated proper nanosize, quasi-spherical shape and improved nuclease protection over naked Chol-siRNA. It was also interesting to note that rHDL provided a highly effective approach to transfer Chol-siRNA across the membrane directly into the cytoplasm via the scavenger receptor BI (SR-BI)-mediated non-endocytotic mechanism, thereby bypassing endo-lysosomal trapping. We also showed clear evidence that the in vitro implementation of rHDL for Chol-siRNA-VEGF (Chol-siRNA targeting vascular endothelial growth factor gene) delivery markedly promoted RNA interference (RNAi)-mediated degradation of VEGF mRNA, resulting in down-regulation of secreted VEGF protein. In vivo fluorescence imaging indicated that near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) displayed long circulation time, SR-BI positive tumor-selective targeting, and efficient cytosolic delivery capabilities. Furthermore, intravenous administration of Chol-siRNA-VEGF-loaded rHDL nanoparticles (rHDL/Chol-siRNA-VEGF complexes) significantly enhanced anti-tumor efficacy against breast cancer, decreased VEGF expression level, and inhibited formation of intratumoral microvessels at the tumor tissue. It was concluded that rHDL possessed therapeutic potential and versatility in mediating Chol-siRNA-VEGF direct cytosolic delivery for target-specific anti-angiogenic therapy in breast cancer.

Wnt3a promotes epithelial-mesenchymal transition, migration, and proliferation of lens epithelial cells.

PURPOSE: Posterior capsular opacification (PCO) is caused mainly by the epithelial-mesenchymal transition (EMT), proliferation, and migration of human lens epithelial (HLE) cells. wingless (Wnt) signaling has been implicated in the fibrotic process by inducing EMT and increasing the proliferation of epithelial cells. This study investigated the role of Wnt3a in PCO formation. METHODS: Wnt3a was overexpressed in the HLE B-3 cell line by transfected Wnt3a-pcDNA3 plasmid. The expressions of Wnt/ß-catenin signaling component proteins, including ß-catenin, E-cadherin, fibronectin, c-Myc, and cyclin D1, were detected by western blot analysis and immunocytofluorescence to confirm the efficiency of transfection efficiency and analyze the effects of overexpression. HLE migration ability was evaluated by transwell migration and wound healing assays, whereas HLE proliferation was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay and flow cytometry. RESULTS: Overexpression of Wnt3a resulted in upregulated expression of ß-catenin, c-Myc, and cyclin D1. Expression of the lens epithelial marker E-cadherin was down-regulated in Wnt3a-overexpressing HLE B-3 cells, whereas that of the mesenchymal marker fibronectin was upregulated. In addition, the morphology of HLE B-3 cells changed from the classic spindle shape to an irregular form. Overexpression of Wnt3a could enhance the ability of migration as determined by transwell migration and wound healing assays as well as promoted the proliferation of HLE B-3 cells by MTT assay and flow cytometry analysis. CONCLUSIONS: Wnt3a can induce EMT, migration, and proliferation of HLE cells and may be a valuable therapeutic target for the prevention and treatment of PCO.